The extracellular matrix is a pre-stressed mechanical network composed of a heterogeneous mixture of modular proteins and oligosaccharides that form the mechanical scaffold of living tissues. It plays crucial roles in the development of the cellular architectures that form all living organisms. A characteristic feature of the extracellular matrix is that its constituent molecules function under a stretching force. Our long-term aim is to understand the mechanisms by which these modular proteins respond and equilibrate with a stretching force, at the single molecule level. Studying the mechanical activity of these proteins will provide a mechanistic understanding of their function in healthy and diseased states. Single molecule studies of the protein modules that compose extracellular matrix proteins such as tenascin and fibronectin have measured their mechanical stability, revealing strong unfolding hierarchies in their mechanical design. However, resistance to unfolding is not sufficient to describe the function of these proteins, which must equilibrate mechanically against a pulling force. This equilibration involves a dynamic balance of folding and unfolding events taking place against the pulling force. The molecular mechanisms underlying this kinetic equilibrium are unknown. We will take advantage of recent advances in single molecule force spectroscopy that now permit a detailed observation of the folding and unfolding kinetics of a protein, while being pulled by a constant mechanical force. Force-clamp spectroscopy combined with protein engineering will be used to study the dynamics of unfolding of the cell binding module of the type III region of fibronectin, 10FNIII, and other key modules of extracellular matrix proteins. We will use the force-quench mode of this technique to study the mechanisms of protein folding under a stretching force. We will use single molecule techniques to study the mechanical strength of engineered disulphide bonds and their effect on the folding/unfolding pathways of selected modules from the proteins fibrillin and fibronectin. Our studies will reveal the molecular mechanisms underlying the dynamic equilibration of proteins with a pulling force, and more generally, demonstrate a novel approach to study the mechanisms of protein folding.